For hundreds of years most drugs were highly impure mixtures of composition derived primarily from plant or animal origin. As recently as the 1920's most active ingredients were used in only partially purified forms. Since then, vastly improved tools and methods for the purification of chemical compounds have been developed enabling identification of compounds that produce beneficial effects. This field science has become known generally as Natural Products Chemistry.
The foundation of Natural Products Chemistry rests on extraction, isolation and purification strategies. As is well appreciated in the art, different isolation procedures oftentimes yield a different profile of chemical compounds. Seemingly minor changes to an isolation procedure such as changing a solvent, the ratio of solvent or even the type of filter paper can result in large changes in the type, amount and purity of chemical compounds obtained. One procure designed to yield large quantities of a crystalline compound, might inadvertently eliminate or inactivate an even more valuable compound in the first extraction step.
A family of compounds isolated from the very slow growing yew (genus Taxus, family Taxaceae), have gained notoriety since the discovery that Taxol was found to be an effective cancer chemotherapeutic agent and was approved by the FDA for treatment of ovarian carcinoma. Since the recognition of Taxol's anticancer activities, research efforts to isolate other compounds from trees of the Genus Taxus have intensified to find improved methods of purification, and synthetic procedures.
Today, the taxane family of terpenes are considered as an exceptionally promising group of cancer chemotherapeutic agents. At least 60 different compounds have been reported in the literature posessing a taxane nucleus (4,8,12,15,15-pentamethyltricyclo[9.3.1.0.sup.3.8 ] pentadecane). ##STR1##
Many taxane derivatives, including taxol, taxotere, and cephalomannine are highly cytotoxic and have been shown to be effective against leukemia, advanced breast and ovarian cancers in clinical trials (W. P. MacGuire et al., Annals of Internal Medicine, vol 111, pg. 273, 1989). They have also exhibited promising activity against a number of other tumor types in preliminary investigations. Taxanes are believed to exert their antiproliferative effect on taxane sensitive cells by inducing tubulin polymerization, thereby forming extremely stable and nonfunctional microtubules (Eric K. Roxinsky et al., Journal of the National Cancer Institute, Vol. 82:1247-1259, 1990).
A major problem with all of the clinical studies is the limited availability of taxanes. For example, the only available natural source for taxol to date are several species of a slow growing Yew (genus Taxus), wherein Taxol is only found in very low concentrations (less than 400 parts per million) in the bark of these trees. Furthermore the extraction is difficult, the process is expensive and the yield of taxol is low (Huang et al., J. Nat. Prod. 49 665 1986 reported a yield of 0.01% taxol from Taxus brevifolia bark).
The number of patents describing the isolation and purification of taxol and taxanes from Taxus bark is increasing.
The procedures currently known for isolating taxol are very difficult and low-yielding. For example, a yield of 0.01% was reported from a large scale isolation starting with at least 806 lbs of Taxus brevifolio bark (Huang et al., J. Nat. Prod., 49:665, 1986). The isolation of taxol was described by other workers: Miller et al., J Org. Chem., 46:1469, 1981; McLaughlin et al., J. Nat. Prod., 44:312, 1981; Kingston et al., J. Nat. Prod., 45:466, 1982, and Senih et al., J. Nat. Prod., 47:131, 1994, U.S. Pat. No. 5,407,674 and U.S. Pat. No. 5,380,916. The reported yields of taxol from various species of yew range from 50 mg/kg to 165 mg/kg (i.e., 0.005-0.017%).
Koppaka (U.S. Pat. No. 5,380,916) describes a method for isolating taxol and its analogues from a crude extract of Taxus brevifolia and Taxus floridana, charactized by treating the crude extract by reverse phase liquid chromatography on an adsorbant, and recovering a number of compounds in pure form by elution. However, reverse phase chromatographic separation of impure taxanes from plant materials is expensive because of the cost of the column materials. Generally reverse phase separation can be used on the crude extraction from bark of some of the Taxus species because of the relatively low concentration of pigments, lipids and waxes and high concentration of taxol; however, the needles tend to contain lesser amounts of taxol and significant amounts of impurities and thus reverse phase chromatography for separation of taxanes form early stages of purification is not practical.
EsSohly et al. (see U.S. Pat. No. 5,480,639), describe methods of obtaining taxanes, comprising extracting and purifying a number of taxanes from ornamental cultivars using a series of organic and aqueous solvents and normal phase chromatography.
Methods of synthesis for the taxane ring skeleton are difficult, producing compounds deficient in pharmacological activity and are currently more expensive than isolation from the plant material. Thus, despite low yields, it is likely that the Taxus plant will remain a predominant reliable supply source for clinical quantities of taxol and its related compounds for years to come.
Although the use of taxol is successful against a number of specific tumor types, it is not universally effective. Hence, there is an urgent need for novel compounds from the taxane family which are closely related to taxol in their chemical structures but with more potent chemotherapeutic activities. New isolation procedures will lead to the purification and identification of new compounds. Moreover, a need exists to simplify the current procedures to produce taxanes and reduce the cost of such production by using simplified extraction and chromatographic techniques.
Therefore, purification techniques which provide high yields of known taxanes and new taxanes are needed to provide greater quantities of these promising therapeutic agents. The present invention provides a purification technique which accomplishes these goals.